R ES E A RC H

◥ synaptic rabies virus revealed the neural

RESEARCH ARTICLE SUMMARY circuit’s synapse. The neural circuit was re-
capitulated in vitro by using nodose neurons
cocultured with either minigut organoids or
NEUROSCIENCE
purified enteroendocrine cells. This system,
coupled to optogenetics and whole-cell patch-
A gut-brain neural circuit for nutrient clamp recording, served to determine the
speed of transduction. Whole-nerve electro-

sensory transduction physiology, along with optical excitation

Melanie Maya Kaelberer, Kelly L. Buchanan, Marguerita E. Klein, Bradley B. Barth,
Marcia M. Montoya, Xiling Shen, Diego V. Bohórquez*
INTRODUCTION: In 1853, Sydney Whiting
wrote in his classic Memoirs of a Stomach,
“…and between myself and that individual
Mr. Brain, there was established a double set
of electrical wires, by which means I could,
with the greatest ease and rapidity, tell him all
enteroendocrine cells and a cranial nerve has
been described. The cells are thought to act on
nerves only indirectly through the slow endo-
crine action of hormones, like cholecystokinin.
Despite its role in satiety, circulating concen-
trations of cholecystokinin peak only several
and silencing, helped to uncover the neuro-
transmission properties of the circuit in vivo.
The underlying neurotransmitter was re-
vealed by using receptor pharmacology and
a fluorescent reporter called iGluSnFR.
RESULTS: Single-cell analyses showed that
a subset of enteroendocrine cells contains
presynaptic adhesion proteins, including
some necessary for synaptic adhesion. Mono-
synaptic rabies tracing

Downloaded from http://science.sciencemag.org/ on September 20, 2018

◥

the occurrences of the day as they arrived, and
he also could impart to me his own feelings
and impressions.” Historically, it is known that
the gut must communicate with the brain, but
the underlying neural circuits and transmitters
mediating gut-brain sensory transduction still
remain unknown. In the gut, there is a single
layer of epithelial cells separating the lumen
from the underlying tissue. Dispersed within
this layer reside electrically excitable cells
termed enteroendocrine cells, which sense in-
gested nutrients and microbial metabolites.
Like taste or olfactory receptor cells, entero-
endocrine cells fire action potentials in the
presence of stimuli. However, unlike other sen-
sory epithelial cells, no synaptic link between
minutes after food is ingested and often after
the meal has ended. Such a discrepancy sug-
gests that the brain perceives gut sensory cues
through faster neuronal signaling. Using a
mouse model, we sought to identify the under-
pinnings of this neural circuit that transduces
a sense from gut to brain.
RATIONALE: Our understanding of brain
neural circuits is being propelled forward by
the emergence of molecular tools that have
high topographical and temporal precision.
We adapted them for use in the gut. Single-
cell quantitative real-time polymerase chain
reaction and single-cell Western blot enabled
the assessment of synaptic proteins. A mono-
ON OUR WEBSITE
revealed that enteroendo-
Read the full article crine cells synapse with
at http://dx.doi. vagal nodose neurons.
org/10.1126/ This neuroepithelial cir-
science.aat5236 cuit connects the intes-
..................................................
tinal lumen with the
brainstem in one synapse. In coculture, this
connection was sufficient to transduce a
sugar stimulus from enteroendocrine cells
to vagal neurons. Optogenetic activation
of enteroendocrine cells elicited excitatory
postsynaptic potentials in connected nodose
neurons within milliseconds. In vivo record-
ings showed that enteroendocrine cells are
indeed necessary and sufficient to trans-
duce a sugar stimulus to the vagus. By using
iGluSnFR, we found that enteroendocrine
cells synthesize the neurotransmitter glu-
tamate, and pharmacological inactivation
of cholecystokinin and glutamate receptors
revealed that these cells use glutamate as a
neurotransmitter to transduce fast, sensory
signals to vagal neurons.

CONCLUSION: We identified a type of gut

sensory epithelial cell that synapses with vagal
neurons. This cell has been referred to as the
gut endocrine cell, but its ability to form a
neuroepithelial circuit calls for a new name.
We term this gut epithelial cell that forms
synapses the neuropod cell. By synapsing with
the vagus nerve, neuropod cells connect the
gut lumen to the brainstem. Neuropod cells
transduce sensory stimuli from sugars in
milliseconds by using glutamate as a neuro-
transmitter. The neural circuit they form gives
the gut the rapidity to tell the brain of all the
occurrences of the day, so that he, too, can
The neuropod cells. (Top left) Neuropod cells synapse with sensory neurons in the small
intestine, as shown in a confocal microscopy image. Blue indicates all cells in villus; green indicates
make sense of what we eat.
▪
green fluorescent protein (GFP) in neuropod cell and sensory neurons. (Bottom left) This neural
circuit is recapitulated in a coculture system between organoids and vagal neurons. Green The list of author affiliations is available in the full article online.
*Corresponding author. Email: diego.bohorquez@duke.edu
indicates GFP in vagal neuron; red indicates tdTomato red fluorescence in neuropod cell. (Right) Cite this article as M. M. Kaelberer et al., Science 361,
Neuropod cells transduce fast sensory signals from gut to brain. Scale bars, 10 µm. eaat5236 (2018). DOI: 10.1126/science.aat5236

Kaelberer et al., Science 361, 1219 (2018) 21 September 2018 1 of 1

R ES E A RC H

◥ either enter the bloodstream or act on nearby

RESEARCH ARTICLE nerves minutes to hours after ingesting a meal
(5). But enteroendocrine cells have several fea-
tures of epithelial transducers: They have mechan-
NEUROSCIENCE ical (6), olfactory (7), and taste (8) receptors;
their membranes contain voltage-gated ion chan-

A gut-brain neural circuit for nutrient nels that render them electrically excitable (9);
and they are capable of forming synapses (10).
Almost two-thirds of enteroendocrine cells syn-
sensory transduction apse with adjacent nerves in the intestinal and
colonic mucosa (10). Similar features have been
confirmed in a subset of colonic enteroendocrine
Melanie Maya Kaelberer1, Kelly L. Buchanan2, Marguerita E. Klein1, Bradley B. Barth3,
cells known as enterochromaffin (11). Therefore,
Marcia M. Montoya3, Xiling Shen3, Diego V. Bohórquez1,4,5* we hypothesized that enteroendocrine cells syn-
apse with the vagus to transduce a sense from
The brain is thought to sense gut stimuli only via the passive release of hormones. This is gut to brain.
because no connection has been described between the vagus and the putative gut epithelial
sensor cell—the enteroendocrine cell. However, these electrically excitable cells contain several Innervated epithelial sensors in the gut
features of epithelial transducers. Using a mouse model, we found that enteroendocrine cells Using mass spectroscopy (see methods and table
synapse with vagal neurons to transduce gut luminal signals in milliseconds by using glutamate S1), we confirmed that enteroendocrine cells ex-
as a neurotransmitter. These synaptically connected enteroendocrine cells are referred to press multiple neuropeptides (12, 13), including

Downloaded from http://science.sciencemag.org/ on September 20, 2018

henceforth as neuropod cells. The neuroepithelial circuit they form connects the intestinal both cholecystokinin (CCK) and peptide YY (PYY).
lumen to the brainstem in one synapse, opening a physical conduit for the brain to sense gut Thus, we identified these cells using CCK and
stimuli with the temporal precision and topographical resolution of a synapse.

W
hereas touch, sight, sound, scent, and
taste are transduced to the brain by in-
nervated epithelial sensor cells (1), per-
ception of gut stimuli is thought to occur
only indirectly, through the slow action
of hormones (2). The putative gut epithelial sen-
sor cells—enteroendocrine cells—are assumed to
lack synapses with the cranial nerve that inner-
vates the viscera—the vagus (3).
Coined in the 1930s (4), the term enteroendo-
crine is rooted in the notion that nutrients stim-
ulate the release of hormones. These neuropeptides
1
Department of Medicine, Duke University, Durham, NC, USA.
2
School of Medicine, Duke University, Durham, USA, NC.
3
Department of Biomedical Engineering, Duke University,
Durham, NC, USA. 4Department of Neurobiology, Duke
University, Durham, NC, USA. 5Duke Institute for Brain
Sciences, Duke University, Durham, NC, USA.
*Corresponding author. Email: diego.bohorquez@duke.edu

Fig. 1. Enteroendocrine cells

contact sensory nerve fibers.
(A) CckGFP_Pgp9.5GFP mice express
GFP in CCK-enteroendocrine cells
and Pgp9.5 sensory nerve fibers.
The two cell types are shown in
the enlarged view, with the CCK-
enteroendocrine cell represented by
a triangle. (B) Confocal microscopy
image of proximal small intestine
villus showing a GFP-labeled
CCK-enteroendocrine cell and
GFP-labeled Pgp9.5 nerve fibers;
18.9 ± 2.0% SEM of CckGFP cells
contact Pgp9.5 fibers (n = 3 mice,
>100 cells per mouse). (C) PYY-stained
enteroendocrine cells (left, green)
in the colon contact Phox2b vagal
nerve fibers (center, red) in a
Phox2bCRE_tdTomato mouse;
merged image is shown on the right.
(D) Two-thirds of CckGFP (green)
enteroendocrine cells colocalize
with the presynaptic marker synapsin-
1 (purple) (n = 6 mice, 200 cells per
mouse). (E) Real-time quantitative
polymerase chain reaction (qPCR)
expression levels of presynaptic
transcripts, including genes encoding for
synaptic adhesion proteins (n = 3 mice,
>10,000 cells per cell type per mouse;
error bars indicate mean ± SEM; a.u.,
arbitrary units; EEC, enteroendocrine cell).
All scale bars, 10 mm.

Kaelberer et al., Science 361, eaat5236 (2018) 21 September 2018 1 of 8

R ES E A RC H | R E S EA R C H A R T I C LE

Downloaded from http://science.sciencemag.org/ on September 20, 2018

Fig. 2. Enteroendocrine cells of the colon and small intestine syn-
apse with vagal nodose neurons. (A) Model of DG-rabies-GFP enema
delivery. (B) PYY cells expressing tdTomato (top left, red) are infected by
DG-rabies-GFP (top right, green). Overlay (bottom) shows overlap of
87.8 ± 2.4% SEM (n = 5 mice). In the absence of G glycoprotein (DG),
DG-rabies-GFP does not spread beyond the infected PYY cell.
(C) EnvA-DG-rabies-GFP virus enters cells via the TvA receptor and
spreads by using the rabG protein within specific cells. (D) EnvA-DG-
rabies-GFP (top right, green) infects PYY cells (top left, red) and spreads
synaptically to underlying colon nerve fibers. Three-dimensional reconstruc-
tion (bottom) shows EnvA-DG-rabies-GFP–infected PYY cell and mono-
synaptically labeled nerve fiber. (E) EnvA-DG-rabies-GFP enema infects
colonic enteroendocrine cells and spreads onto vagal neurons in the nodose
ganglion (green). (F) In additional experiments, DG-rabies-GFP delivered
by oral gavage spreads in the intestinal lumen of CckCRE_rabG-TvA mice to
label the nucleus tractus solitarius (green). This neuroepithelial circuit links
the intestinal lumen with the brainstem. The inset shows the location of the
nucleus tractus solitarius in the mouse brain. All scale bars, 10 mm.

PYY. In the mouse small intestine and colon,
enteroendocrine cells contacted sensory nerve
fibers (Fig. 1, A to C). About one in five CCK-
expressing enteroendocrine (CCK-enteroendocrine)
cells contacted Pgp9.5 sensory nerve fibers that
express green fluorescent protein (Pgp9.5GFP
nerve fibers) (18.9 ± 2.0% SEM, >100 cells per
mouse, n = 3 mice) (Fig. 1B). CCK-enteroendocrine
cells immunoreact with an antibody against
the presynaptic protein synapsin-1 (Fig. 1D),
showing that these connections have synaptic
features. Furthermore, using single-cell Western
blot, we found that 83% of enteroendocrine cells
contain synapsin-1 (164 of 198 CckGFP cells an-
alyzed) (fig. S1). Compared with other intestinal
epithelial cells, purified CCK-enteroendocrine
cells express the synaptic adhesion genes Efnb2,
Lrrtm2, Lrrc4, and Nrxn2 (Fig. 1E), showing
that these epithelial sensors have the machin-
ery to form synapses.
From gut lumen to brainstem
in one synapse
To determine the source of neurons synapsing
with enteroendocrine cells, we used a modified
rabies virus (DG-rabies-GFP) (10). This rabies virus
infects neurons but lacks the G glycoprotein nec-
essary for transsynaptic spread (Fig. 2A) (14).
In intestinal organoids, rabies prefers to infect
enteroendocrine cells over other epithelial cells
(fig. S2A). In the mouse, when introduced into
the lumen of the colon by enema, almost 9 out of
10 infected cells are PYY-enteroendocrine cells
(87.8 ± 2.4% SEM, n = 5 mice) (Fig. 2B) (10). The
lack of fluorescence in the underlying mucosa
shows that, in the absence of its G glycoprotein,
the rabies virus does not spread beyond infected
enteroendocrine cells.
To trace the neural circuit, we bred a mouse
(strain PyyCRE_rabG-TvA) in which enteroendo-
crine cells express the G glycoprotein (rabG) (Fig.
2C). In these mice, rabies delivered by enema
infects enteroendocrine cells and spreads through
synapses onto nerves. Some of the nerve fibers
can be traced to vagal nodose neurons (control
group: 0 positive out of 3 PyyCRE_tdTomato
mice; experimental group: 4 positive out of
5 PyyCRE_rabG-TvA mice). Furthermore, an
enema of the chemical tracer dye Fast Blue
labeled both nodose ganglia, confirming that
the vagus indeed innervates the distal colon (15).
In control experiments in which the right cer-
vical vagus was severed, the Fast Blue enema
labeled the left (intact) but not the right (vagot-
omized) nodose (fig. S3).
Because DG-rabies-GFP can infect any neuro-
nal cell it contacts, we restricted its entrance to
enteroendocrine cells only by using an EnvA-
coated rabies (EnvA-DG-rabies-GFP) (Fig. 2C).
EnvA is an envelope glycoprotein of the avian
sarcoma leukosis virus that binds to the avian

Kaelberer et al., Science 361, eaat5236 (2018) 21 September 2018 2 of 8

R ES E A RC H | R E S EA R C H A R T I C LE

Downloaded from http://science.sciencemag.org/ on September 20, 2018

Fig. 3. Enteroendocrine cells transduce glucose stimuli onto vagal neu-
rons. (A) Model of intestinal intraluminal perfusion and vagal nerve
electrophysiology. (B) Normalized traces for baseline, Ensure, 300 mM sucrose,
and 300 mM sucrose with 3 mM phloridzin (phl) in wild-type mice. Gray bar
indicates treatment period; shading indicates SEM. (C) Ensure, 300 mM
sucrose, and 150 mM D-glucose stimulate vagal firing rate, which is abolished
by SGLT1-blocker phloridzin [n ≥ 5 mice; *P < 0.0001, analysis of variance
(ANOVA) with post hocTukey’s HSD test; error bars indicate SEM]. (D) Intestinal
epithelial cells express Sglt1, but nodose neurons do not (n = 3 mice,
>10,000 cells per cell type per mouse; data are presented as mean ± SEM).
(E) Nodose neurons cultured alone for electrophysiology (widefield microscopy
image on left, model on right). (F) Nodose neurons do not respond to 10 mM
glucose in voltage-clamp (left trace) or current-clamp (right trace) mode. Insets
show that neurons respond to voltage or current pulse, indicating viability.
(G) Nodose neurons cocultured with GFP-positive enteroendocrine cells for
electrophysiology (image on left, model on right). Innervated enteroendocrine
cells are shown at the bottom. (H) In coculture, glucose evoked EPSCs
(top left) and action potentials (top right) in connected neurons (scale of current
or voltage and time are shown below the traces). Dashed-line box indicates
action potentials expanded in right inset. Quantification of EPSC amplitude
and frequency (bottom left and center; n = 21 neurons alone; n = 6 neurons
connected to enteroendocrine cells) and action potentials (bottom right;
n = 21 alone; n = 5 neurons connected to enteroendocrine cells) in
GFP-negative (–) and -positive (+) cells. All scale bars, 10 mm.

TvA receptor. Therefore, EnvA-DG-rabies-GFP
only infects cells that express the TvA re-
ceptor. In the PyyCRE_rabG-TvA mouse, PYY-
enteroendocrine cells express the TvA receptor,
and an enema of EnvA-DG-rabies-GFP infects
enteroendocrine cells exclusively. Then, it spreads
to synaptically connected neurons. Of a total of
nine mice, five had visible infection of nerve fibers
in the colon (Fig. 2D), and two of those five had
visible infection in the vagal nodose (Fig. 2E and
movie S1; confirmed in vitro in fig. S4). Labeled
fibers were also observed in the dorsal root ganglia
of four out of the five infected mice (fig. S5). No
infection of nerves was observed in littermate con-
trols that lack CRE recombinase (n = 5 mice).

Kaelberer et al., Science 361, eaat5236 (2018) 21 September 2018 3 of 8

R ES E A RC H | R E S EA R C H A R T I C LE

Downloaded from http://science.sciencemag.org/ on September 20, 2018

Fig. 4. Millisecond transduction from enteroendocrine cells to vagal
neurons. (A) Model of intraluminal photostimulation and vagal
electrophysiology. (B) In CckCRE_ChR2-tdTomato mice, intestinal
enteroendocrine cells express ChR2. (C) Normalized traces for 473-nm
intraluminal laser, 300 mM sucrose, and baseline in CckCRE_ChR2 mice.
Shading indicates SEM. (D) 473-nm intraluminal laser stimulates vagal firing
rate in CckCRE_ChR2, but not wild-type, mice (n ≥ 5 mice; *P < 0.05, ANOVA
with post hoc Tukey’s HSD test; error bars indicate SEM). (E) Patch-clamp
electrophysiology of neurons (model on left) in coculture with CckCRE_ChR2
cells (image on right). (F) In coculture, 473-nm photostimulation evoked
EPSCs (trace on left) in connected nodose neurons (quantification on right)
(n = 9 neurons connected to enteroendocrine cells; –, neurons alone; +,
neurons cocultured with enteroendocrine cells; DT, time between stimulus
and onset of EPSCs). Scale of current and time is shown below the trace.
(G) Model of intraluminal photoinhibition and vagal electrophysiology.
(H) In CckCRE_Halo-YFP mice, intestinal enteroendocrine cells express
halorhodopsin (eNpHR3.0). (I) Normalized traces for baseline, 300 mM
sucrose, and 300 mM sucrose with 532-nm intraluminal laser. Shading
indicates SEM. (J) In CckCRE_Halo, but not wild-type, mice, a 532-nm
intraluminal laser abolishes the effect of sucrose on vagal firing rate
(n ≥ 5 mice per group; *P < 0.0001, ANOVA with post hoc Tukey’s HSD test;
error bars indicate SEM). All scale bars, 10 mm.

Delivering the virus by oral gavage into
CckCRE_rabG-TvA mice yielded similar results
(fig. S5). In these mice, labeled vagal nodose neu-
rons projected upstream into the nucleus tractus
solitarius of the brainstem (Fig. 2F). Monosyn-
aptic rabies tracing shows a neural circuit link-
ing the small intestine or colon lumen to the
brainstem in one synapse.
A gut-brain neural circuit in a dish
In coculture, vagal nodose neurons clearly ex-
tended axons to enteroendocrine cells of intestinal
organoids (fig. S4A and movie S2). We traced this
neural circuit in vitro using EnvA-DG-rabies-GFP
to confirm that synapses are formed. To ensure
that only infected neurons spread EnvA-DG-
rabies-GFP, nodose neurons were incubated with
virus before coculture with organoids. In control
experiments, EnvA-DG-rabies-GFP did not infect
wild-type nodose neurons (fig. S4B). However,
EnvA-DG-rabies-GFP infected vagal nodose neu-
rons that express the TvA receptor (Phox2bCRE_
rabG-TvA). Forty-eight to 72 hours after coculture,
the virus spread onto enteroendocrine cells in
intestinal organoids, demonstrating synaptic con-
nection in vitro (fig. S4C).
Transduction of a sense from gut to brain
We tested the function of this neuroepithelial
circuit using luminal stimuli and whole-nerve
electrophysiology. The initial stimulus used was
Ensure—a whole-nutrient solution. Luminal En-
sure stimulated an increase in vagal firing rate
(Fig. 3, A to C). Next, we focused on a distinctive
nutrient, sugar. When ingested, sugar is sensed
in the duodenum, but it is unclear whether this
stimulus is sensed by the vagus directly or trans-
duced via enteroendocrine cells (16). In wild-
type mice, perfusing the sugar sucrose (100 to
300 mM) significantly increased vagal firing rate

Kaelberer et al., Science 361, eaat5236 (2018) 21 September 2018 4 of 8

R ES E A RC H | R E S EA R C H A R T I C LE

Downloaded from http://science.sciencemag.org/ on September 20, 2018

Fig. 5. Glutamate is used as a neurotransmitter between enteroendo-
crine cells and neurons. (A) Model of synaptic neurotransmission in
enteroendocrine cells. (B) Enteroendocrine cells express the vesicular
glutamate genes encoding VGLUT1 and 2 (Slc17a7 and Slc17a6)
(quantification by qPCR on left, confocal microscopy images on right).
(C) CckCRE_tdTomato enteroendocrine cells were cocultured with HEK cells
that express the glutamate sniffer protein, iGluSnFR (multiphoton microscopy
image on left, model on right). (D) A stimulus of 40 mM D-glucose
administered during the time period indicated by the beige shading elicits
a response in iGluSnFR-HEK cells (n = 3 cultures; individual cell, gray trace;
average of all cells, black trace). DF/F, difference in fluorescence intensity
between resting state and after stimulus. (E) Coculture with neurons
and CckCRE_ChR2 cells (multiphoton microscopy image on left) for
electrophysiology of neurons and microperfusion of the glutamate-receptor
blocker kynurenic acid (model on right). (F) In coculture, 473-nm photo-
stimulation evoked EPSCs in connected nodose neurons, these currents were
abolished, and no response was observed with the addition (+) of 3 mM
kynurenic acid. The response was recovered after the drug was washed
off (indicated by second “–” condition on right) (n = 4 neurons connected
to enteroendocrine cells). All scale bars, 10 mm.

over baseline (Fig. 3, B and C, and fig. S6).
D -Glucose (150 mM), but not fructose (150 mM),
had the same effect. No effect was observed when
the vagus was severed (fig. S7), when hyper-
osmolar phosphate-buffered saline was perfused
(700 mosmol), or when sucrose was applied
intraperitoneally (300 mM) (fig. S8). The vagal
response was abolished when sucrose was per-
fused with phloridzin, a blocker of the electro-
genic glucose transporter SGLT1 (17) (Fig. 3, B
and C). A transcription profile showed that, un-
like vagal nodose neurons, CCK-enteroendocrine
cells express Sglt1, suggesting that the stimulus is
transduced by the epithelial cells (Fig. 3D).
Evidence gathered on dissociated colonic en-
teroendocrine cells, and the enteroendocrine-like
cell line STC1, has shown that enteroendocrine
cells sense glucose (18). We therefore packaged
a rabies virus to carry the calcium reporter
GCaMP6s (DG-rabies-GCaMP6s) and used it to
infect enteroendocrine cells in intestinal organoids.
When presented with D-glucose (10 mM), calcium
transients were elicited in CCK-enteroendocrine
cells (56.0 ± 20.0% of the KCl control response;
n = 3 cells) (fig. S2, B to D). One previous report
found that rat nodose neurons respond to glu-
cose (19). However, in contrast with enteroendo-
crine cells, vagal neurons are unlikely to face
steep changes in glucose concentrations be-
cause they do not contact the intestinal lumen
(20). We therefore measured calcium transients
in dissociated nodose neurons and found that
D-glucose (10 mM) did not elicit a response
(fig. S9, A and B) (n = 246 cells pooled from
three mice).
To discard the possibility that only nodose
neurons innervating the intestine may sense
glucose, we retrotraced them by injecting Fast
Blue dye into the duodenum (fig. S9C). In Fast
Blue–labeled vagal neurons, no calcium response
was observed in the presence of D-glucose (20 mM)
(fig. S9C). Furthermore, neither excitatory currents
nor action potentials were observed in the pres-
ence of a D-glucose (10 to 20 mM) stimulus
using patch-clamp electrophysiology (Fig. 3, E
and F). Current injection demonstrated that these
cultured nodose neurons were functionally viable
(inset of Fig. 3F).

Kaelberer et al., Science 361, eaat5236 (2018) 21 September 2018 5 of 8

R ES E A RC H | R E S EA R C H A R T I C LE

We then cocultured vagal nodose neurons

with intestinal enteroendocrine cells (10). After
48 to 72 hours, there were visible connections
between neurons and enteroendocrine cells (Fig.
3G). Coculturing did not alter the resting mem-
brane potential, the current, or the spike thresh-
old of the vagal nodose neurons. However, a
D -glucose (10 mM) stimulus now evoked excit-
atory postsynaptic currents (EPSCs) and action
potentials in those neurons connected to entero-
endocrine cells (Fig. 3H). In voltage-clamp mode,
the average current of the EPSCs was 61.65 ±
15.21 pA, and the average frequency was 0.86 ±
0.17 Hz (n = 6 neurons connected to entero-
endocrine cells). In current-clamp mode, this
in vitro connection was sufficient to elicit action
potentials in the connected neurons (average of
2 ± 0.32 action potentials, n = 5 neurons con-
nected to enteroendocrine cells).

Synaptic speed and specificity

Downloaded from http://science.sciencemag.org/ on September 20, 2018

Two recent reports have shown that hypo-
thalamic neurons controlling food intake are
inhibited by nutrients within seconds of the
nutrients entering the duodenum (21, 22). There-
fore, it is likely that enteroendocrine cells trans-
duce sensory signals from nutrients at a much
faster rate than previously thought possible. To
test the speed of transduction, we bred a mouse
(strain CckCRE_ChR2-tdTomato) in which en-
teroendocrine cells express channelrhodopsin 2
(ChR2) —an excitatory light-gated ion channel
activated by 473-nm light (Fig. 4, A and B). A
473-nm stimulus applied to these cells elicited
excitatory currents and significantly reduced food
intake by the mice, showing functional expression
of the channel (fig. S10) (see methods).
Vagal firing rate is significantly increased
when a 473-nm laser stimulus is applied to the
duodenal lumen of CckCRE_ChR2 mice. No
response was observed in wild-type controls
(Fig. 4, C and D; for laser-activation controls,
see fig. S11). The firing rate increased rapidly
after laser stimulation, reaching its peak, on
average, in 72.7 ± 20.9 s (fig. S12). In vitro, vagal
nodose neurons cultured alone did not respond
to photostimulation. To determine the precise
transduction speed, we cocultured them with
CckCRE_ChR2 enteroendocrine cells (Fig. 4E).
In vagal nodose neurons connected to entero-
endocrine cells, a 470-nm photostimulus elicited
EPSCs within 60 to 800 ms (n = 9 pairs) (Fig. 4F).
To test the specificity of transduction, we bred
a mouse (CckCRE_Halo-YFP) in which intestinal
enteroendocrine cells express the light-inhibitory
channel eNpHR3.0 (halorhodopsin)—an inhibi-
tory light-gated ion channel activated by 532-nm
light (Fig. 4, G and H)—and yellow fluorescent
protein (YFP). In these mice, luminal sucrose
(300 mM) elicited a vagal response; however,
when a 532-nm laser stimulus was presented
along with the sucrose, vagal activity was abol-
ished (Fig. 4, I and J; for laser activation con-
trols, see fig. S13). In control wild-type mice, a
532-nm laser stimulus failed to attenuate the
sucrose response. These data revealed that entero-
endocrine cells are necessary and sufficient to
Fig. 6. The rapid vagal response to sucrose is dependent on glutamate, whereas CCK
contributes to the prolonged response. (A) Normalized traces for baseline, 300 mM sucrose,
300 mM sucrose after treatment with 2 mg/kg devazepide, and 300 mM sucrose after treatment
with glutamate inhibitor cocktail KA/AP3 [150 mg/kg kynurenic acid (KA) with 1 mg/kg DL-2-amino-3-
phosphonoproprionic acid (AP-3)] in wild-type mice. Shading indicates SEM. (B) Normalized
traces for baseline, 300 mM sucrose, and 300 mM sucrose after treatment with 150 mg/kg KA in
wild-type mice. Shading indicates SEM. (C) KA/AP3 attenuates the maximum normalized vagal
firing rate in response to sucrose, whereas devazepide and KA alone do not. (D) KA/AP3 and
KA alone prolong the time to peak from an average of 92.8 s to 198 and 179 s, respectively.
Devazepide (2 mg/kg) does not significantly change the time to peak (mean = 67.1 s). For (C) and
(D), n ≥ 5 mice per group; *P < 0.05, ANOVA with post hoc Tukey’s HSD test; error bars indicate SEM.
transduce a glucose stimulus onto vagal neurons 3 mice). Moreover, vagal nodose neurons express
within milliseconds. at least eight glutamate receptors (fig. S14).
To test whether enteroendocrine cells release
The neurotransmitter glutamate, we used the sniffer protein iGluSnFR.
The possibility exists that innervated enteroen- This membrane-bound protein fluoresces green
docrine cells could use a classic neurotransmitter in the presence of glutamate (27). Transfected
to transduce the above-described sensory signals. iGluSnFR–HEK (human embryonic kidney) cells
Other sensory epithelial transducers—including did not respond to a D-glucose (40 mM) stim-
photoreceptors (23), auditory hair cells (24), ulus but did respond to glutamate (100 mM)
Merkel cells (25), and olfactory receptor cells (26)— (fig. S15). We then cocultured iGluSnFR-HEK
use vesicular glutamate as a neurotransmitter. cells with Tomato-expressing enteroendocrine
Thus, we hypothesized that enteroendocrine cells (CckCRE_tdTomato) (Fig. 5C). This time,
cells use glutamate as a neurotransmitter as well. when presented with a D-glucose stimulus
We found that intestinal enteroendocrine cells (40 mM), iGluSnFR-HEK cells fluoresced green
express significant quantities of the transcript (n = 3 cultures; Fig. 5D), indicating that entero-
for the vesicular glutamate transporter 1 pro- endocrine cells release glutamate. Then, we
tein (VGLUT1) (Fig. 5, A and B). In a transgenic cocultured CckCRE_ChR2 enteroendocrine cells
Vglut1CRE_YFP mouse, fluorescence was ob- with vagal neurons to determine if glutamate
served in distinct intestinal epithelial cells that serves as a neurotransmitter in this synapse. In
resemble enteroendocrine cells, and almost 4 in connected neurons, a 470-nm stimulus elicited
10 of those fluorescent cells costained for CCK EPSCs that were abolished by adding kynurenic
(38.80 ± 2.53% SEM, 100 cells per mouse, n = acid (3 mM), an ionotropic glutamate-receptor

Kaelberer et al., Science 361, eaat5236 (2018) 21 September 2018 6 of 8

R ES E A RC H | R E S EA R C H A R T I C LE
blocker (Fig. 5, E and F). The response was re-
covered once the blocker was washed away (n =
4 neurons connected to enteroendocrine cells)
(Fig. 5F).
Hormone versus neurotransmitter
In a transgenic mouse in which VGLUT1-
enteroendocrine cells express ChR2 (Vglut1CRE_
ChR2-YFP), a luminal laser stimulus of 473-nm
significantly increased vagal firing rate (fig. S16).
The amplitude and timing of the peak response
was comparable to the CckCRE_ChR2 experi-
ments (figs. S12 and S16). The same laser
stimulus applied to the subdiaphragmatic or
cervical vagus did not alter firing rate (fig. S17).
However, the response was abolished when the
473-nm laser was presented along with a cock-
tail of glutamate-receptor blockers [metabotropic
blocker AP-3 (1 mg per kg of body weight) with
ionotropic blocker kynurenic acid (150 mg/kg)]
(fig. S16). These data revealed a type of entero-
endocrine cell that uses glutamate to drive
vagal firing.
Next, we compared the respective contribu-
tions of CCK and glutamate to vagal firing. The
peak vagal firing rate elicited by a sucrose stim-
ulus was not affected when the CCK-A receptor
was blocked with devazepide (2 mg/kg) (Fig. 6, A
and C). In control experiments, the same dose of
devazepide fully blocked the vagal response to
luminal CCK (fig. S18). Although the peak re-
sponse and time to peak were not altered by
devazepide, the length of the response was at-
tenuated after 120 s (Fig. 6, A, C, and D; and figs.
S18 and S19), suggesting that it takes minutes
for released CCK to stimulate vagal firing. By con-
trast, blocking both ionotropic and metabotropic
glutamate receptors attenuated the speed, peak,
and magnitude of the vagal response to sucrose
(Fig. 6, C and D, and fig. S19). Indeed, the first
60 s of the vagal response to sucrose was sup-
pressed by the ionotropic blocker kynurenic acid
alone (Fig. 6B and fig. S20), delaying the time
to peak to around 180 s (Fig. 6D and fig. S18C).
These data revealed that synaptic glutamate is
used by an epithelial sensor cell in the gut to
rapidly transduce luminal stimuli to the central
nervous system.
The neuropod cells
In recent years, enteroendocrine cells have emer-
ged as sensors of mechanical, chemical, and
bacterial signals in the gastrointestinal tract
(2, 3). However, their transducer properties have
been obscured by their name. By synapsing with
the vagus, these sensor cells provide a neuro-
epithelial circuit for fast sensory transduction.
As such, we see the need for a new name to refer
to gut sensory epithelial cells that synapse with
nerves. We refer to these cells as neuropod cells.
We hypothesize that the gut-brain neural circuit
formed by neuropod cells and vagal nodose neu-
rons could lead to the following possibilities: (i)
rapid computation of stimuli to distinguish their
physical (e.g., volume) versus chemical (e.g., cal-
orie) properties; (ii) precise sensory representation
of specific gastrointestinal regions; (iii) localized
Kaelberer et al., Science 361, eaat5236 (2018) 21 September 2018
plasticity encoded within the neural circuit; and
(iv) timely vagal efferent feedback to modulate
gastrointestinal sensory function. Like other sen-
sory transducers, neuropod cells use synaptic
signals to help the brain make sense of the food
we eat.
Materials and methods summary
Animals
Mouse care and experiments were carried out
in accordance with protocols approved by the
Institutional Animal Care and Use Committee
at Duke University Medical Center under the
protocol A009-16-01. Mice were housed in
the Duke University animal facilities, where
they were kept on a 12-hour light-dark cycle.
They received food and water ad libitum. The
specific strains can be found in the supple-
mental methods.
Rabies production and tracing
G-deleted rabies virus production was performed
in house as described in Wickersham et. al (28).
For colon monosynaptic tracing, P1 mice were
given an enema of EnvA-DG-rabies-GFP (5.9 ×
109 ffu/ml). For small intestine monosynaptic
tracing, P1 mice were given a gavage of DG-
rabies-GFP (9.8 × 108 ffu/ml). Mice were sacrificed
7 days after exposure at P8. Harvested tissue was
fixed in 4% PFA then treated with serial sucrose
solutions. Ganglia were whole-mount imaged
with a multiphoton microscopy system (Bruker
Ultima IV with a Chameleon Vision II tunable
laser). All other tissue was frozen in OCT blocks
and sectioned for immunohistochemistry.
Organoid culture
Organoids were cultured using a protocol adapt-
ed from Sato et al. 2009 (29). Isolated crypts were
resuspended in Matrigel (Corning #356231) and
plated 50 µl per well in a 24-well plate in orga-
noid media. Organoid media contains 1x Glutamax,
10 10mM HEPES, 200 U/ml Penicillin-Streptomycin,
1× N2 supplement, 1× B27 supplement, 0.25 ng/ml
EGF, 50 ng/ml Noggin, and 100 ng/ml r-Spondin in
Advanced DMEM/f12.
Enteroendocrine cell and nodose
neuron coculture
Enteroendocrine cells of CckGFP and CckCRE_
ChR2-tdTomato small intestines were isolated as
previously described in Bohórquez et al. (10).
Enteroendocrine cells were sorted into organoid
culture media (listed above) plus 10 ng/ml NGF.
Sorted cells were plated on 1% Matrigel coated
12-mm coverslips at a concentration of ~5000 to
10,000 enteroendocrine cells per coverslip. No-
dose neurons were dissected and incubated with
Liberase (Roche) digestion enzyme. Neurons in
media were plated evenly on up to eight cover-
slips with enteroendocrine cells. Patch-clamp
electrophysiology was performed 2 to 5 days
after plating.
Immunohistochemistry
Immunohistochemistry was performed as pre-
viously described in Bohórquez et al. (10). Pri-
mary antibodies: Rb-Anti-PYY [DVB3] (1:1000);
Rb-Anti-CCK (1:1000; courtesy of Rodger Liddle
or Phoenix Pharmaceuticals H-069-04); Gt-Anti-
PSD95 (1:500; Santa Cruz Biotechnology: sc-6926);
Rb-Anti-Syn1 (1:500; Cell Signaling Technology:
5297S); Ck-Anti-GFP (1:500; Abcam: ab13970].
Secondary antibodies from Jackson Immuno-
Reseach: Dk-Anti-Rb-488 (1:250); Dk-Anti-
Rb-Cy3 (1:250); Dk-Anti-Gt-Cy5 (1:250); and
Dk-Anti-Ck-488 (1:250). Imaging was done on
a Zeiss 880 Airyscan inverted confocal micro-
scope. Data are presented as the mean percent-
age ± SEM.
Real-time quantitative PCR
RNA from CckGFP-positive and -negative epi-
thelial cells was extracted based on the man-
ufacturer’s protocol using the RNeasy Micro Plus
Kit (Qiagen #74034). Then cDNA was produced
per manufacturer’s protocol using the High Ca-
Downloaded from http://science.sciencemag.org/ on September 20, 2018
pacity cDNA Reverse Transcription Kit (Applied
Biosystems #4368814). TaqMan probes used
are listed in supplemental materials. Real-time
qPCR was run on a StepOnePlus System (Thermo
Fischer), using TaqMan Fast Universal PCR Master
Mix (Applied Biosystems #4352042) according
to the manufacturer’s protocol. Transcription
rate was determined as 2-DCt, or compared as fold-
change over GFP negative epithelial cells using
2-DDCt. All values are reported as mean ± SEM.
Electrophysiology
Enteroendocrine cells and nodose neurons were
cocultured as described above. Recordings were
carried out at room temperature using a Multi-
Clamp 700B amplifier (Axon Instruments), dig-
itized using a Digidata 1550A (Axon Instruments)
interface, and pClamp software (Axon Instru-
ments) for data acquisition. Recordings were
made using borosilicate glass pipettes pulled to
~3.5 MW resistance. Extracellular solution con-
tained (in mM): 140 NaCl, 5 KCl, 2 CaCl2, 2 MgCl2,
10 HEPES, pH 7.4 (300 to 305 mosmol). For
voltage-clamp recordings, intracellular solution
contained (in mM): 140 CsF, 10 NaCl, 0.1 CaCl2,
2 MgCl2, 1.1 EGTA, 10 HEPES, 10 sucrose (pH
7.25, 290 to 295 mosmol). For current-clamp
recordings, intracellular solution contained
(in mM): 140 KCl, 0.5 EGTA, 5 HEPES, 3 Mg-
ATP, 10 sucrose (pH 7.25, 290 to 295 mosmol).
Data are presented as the mean ± SEM, and
significance was determined using a two-tailed
Student’s t test.
iGluSnFR-HEK cell and enteroendocrine
cell coculture and imaging
CckCRE_tdTomato enteroendocrine cells were
isolated as described above. Isolated cells were
mixed with iGluSnFR-HEK cells at a ratio of 10:1,
then plated on 1% Matrigel coated coverslips.
Control iGluSnFR-HEK cells were plated alone.
Cells were incubated for 12 to 18 hours before
imaging. Coverslips were imaged using a multi-
photon microscopy system (Bruker Ultima IV
with a Chameleon Vision II tunable laser). Imag-
ing series were analyzed using Fiji (it’s just
ImageJ), and cell traces were plotted with Excel.
7 of 8
R ES E A RC H | R E S EA R C H A R T I C LE
Vagus nerve recording
Wild-type control (n = 5 to 9), CckCRE_ChR2-
tdTomato (n = 6), CckCRE_Halo-YFP (n = 5),
and Vglut1CRE_ChR2-YFP (n = 6) mice were
used for vagal recordings. The cervical vagus was
exposed in anesthetized mice and two platinum
iridium wires (Medwire by Sigmund Cohn Corp)
were looped around the vagus nerve for record-
ing. A 20-gauge gavage needle was surgically
inserted through the stomach wall and into the
duodenum. Saline and stimulant tubes were
connected to the gavage needle. For optogenetic
experiments, a fiber optic cable (FT020, ThorLabs)
was threaded through the gavage needle into
the lumen of the duodenum. A perfusion exit
incision was made 10 cm distal to the pyloric
sphincter. During each recording, PBS was con-
stantly perfused through the duodenum using a
peristaltic pump (Cole-Parmer) at the lowest
setting for a flow rate of ~400 µl PBS per minute.
For stimulus delivery, see extended methods in
supplemental materials. Data acquisition: A dif-
ferential amplifier and bandpass filter (1000×
gain, 300-Hz to 5-kHz bandpass filter; A-M Sys-
tems LLC) was used and the signal was processed
using a data acquisition board and software
(20-kHz sampling rate; Signal Express, National
Instruments Corp). The raw data was analyzed
using a spike sorting algorithm (MATLAB by
MathWorks). Spikes were detected using simple
threshold detection based on RMS noise. The
firing rate was calculated using a Gaussian kernel
smoothing algorithm (200-ms time scale). Statis-
tical Methods: Stimulation response was quanti-
fied as the maximum firing rate after stimulation
(stimulant conditions) or during recording (base-
line). Time to peak was calculated as time from
start of stimulus to maximum firing rate. Area
under the curve was calculated as area under the
curve for the entire 6-min recording. Maximum
firing rate, time to peak, and area under curve are
analyzed across genotype, stimulation condition,
and their interaction term by ANOVA, followed
by Tukey HSD post hoc testing (JMP by SAS
Institute).
RE FE RENCES AND N OT ES
1. B. Alberts, D. Bray, J. Lewis, M. Raff, K. Roberts, J. D. Watson,
Molecular Biology of the Cell (Garland, ed. 3, 1994),
pp. 907–982.
2. A. Psichas, F. Reimann, F. M. Gribble, Gut chemosensing
mechanisms. J. Clin. Invest. 125, 908–917 (2015). doi: 10.1172/
JCI76309; pmid: 25664852
3. J. B. Furness, L. R. Rivera, H. J. Cho, D. M. Bravo, B. Callaghan,
The gut as a sensory organ. Nat. Rev. Gastroenterol.
Hepatol. 10, 729–740 (2013). doi: 10.1038/nrgastro.2013.180;
pmid: 24061204
4. F. Feyrter, Über diffuse endokrine epitheliale Organe
(J. A. Barth, Leipzig, Germany, 1938).
Kaelberer et al., Science 361, eaat5236 (2018) 21 September 2018
5. J. F. Rehfeld, The new biology of gastrointestinal hormones.
Physiol. Rev. 78, 1087–1108 (1998). doi: 10.1152/
physrev.1998.78.4.1087; pmid: 9790570
6. D. Castaneda et al., Mechanosensitive ION channel
Piezo2 distribution in mouse small bowel and colon
enterochromaffin cells. Gastroenterology 152, S180 (2017).
doi: 10.1016/S0016-5085(17)30916-2
7. T. Braun, P. Voland, L. Kunz, C. Prinz, M. Gratzl,
Enterochromaffin cells of the human gut: Sensors for spices
and odorants. Gastroenterology 132, 1890–1901 (2007).
doi: 10.1053/j.gastro.2007.02.036; pmid: 17484882
8. H. J. Jang et al., Gut-expressed gustducin and taste receptors
regulate secretion of glucagon-like peptide-1. Proc. Natl. Acad.
Sci. U.S.A. 104, 15069–15074 (2007). doi: 10.1073/
pnas.0706890104; pmid: 17724330
9. G. J. Rogers et al., Electrical activity-triggered glucagon-like
peptide-1 secretion from primary murine L-cells. J. Physiol.
589, 1081–1093 (2011). doi: 10.1113/jphysiol.2010.198069;
pmid: 21224236
10. D. V. Bohórquez et al., Neuroepithelial circuit formed by
innervation of sensory enteroendocrine cells. J. Clin. Invest.
125, 782–786 (2015). doi: 10.1172/JCI78361; pmid: 25555217
11. N. W. Bellono et al., Enterochromaffin cells are gut
chemosensors that couple to sensory neural pathways.
Cell 170, 185–198.e16 (2017). doi: 10.1016/j.cell.2017.05.034;
pmid: 28648659
12. A. L. Haber et al., A single-cell survey of the small intestinal
epithelium. Nature 551, 333–339 (2017). doi: 10.1038/
nature24489; pmid: 29144463
13. L. L. Glass et al., Single-cell RNA-sequencing reveals a distinct
population of proglucagon-expressing cells specific to the
mouse upper small intestine. Mol. Metab. 6, 1296–1303 (2017).
doi: 10.1016/j.molmet.2017.07.014; pmid: 29031728
14. N. R. Wall, I. R. Wickersham, A. Cetin, M. De La Parra,
E. M. Callaway, Monosynaptic circuit tracing in vivo through
Cre-dependent targeting and complementation of modified
rabies virus. Proc. Natl. Acad. Sci. U.S.A. 107, 21848–21853
(2010). doi: 10.1073/pnas.1011756107; pmid: 21115815
15. S. M. Altschuler, J. Escardo, R. B. Lynn, R. R. Miselis,
The central organization of the vagus nerve innervating the
colon of the rat. Gastroenterology 104, 502–509 (1993).
doi: 10.1016/0016-5085(93)90419-D; pmid: 8425692
16. E. K. Williams et al., Sensory neurons that detect stretch and
nutrients in the digestive system. Cell 166, 209–221 (2016).
doi: 10.1016/j.cell.2016.05.011; pmid: 27238020
17. A. Sclafani, H. Koepsell, K. Ackroff, SGLT1 sugar transporter/
sensor is required for post-oral glucose appetition. Am. J.
Physiol. Regul. Integr. Comp. Physiol. 310, R631–R639 (2016).
doi: 10.1152/ajpregu.00432.2015; pmid: 26791832
18. F. Reimann et al., Glucose sensing in L cells: A primary cell
study. Cell Metab. 8, 532–539 (2008). doi: 10.1016/
j.cmet.2008.11.002; pmid: 19041768
19. G. Grabauskas, I. Song, S. Zhou, C. Owyang,
Electrophysiological identification of glucose-sensing neurons
in rat nodose ganglia. J. Physiol. 588, 617–632 (2010).
doi: 10.1113/jphysiol.2009.182147; pmid: 20008464
20. H. R. Berthoud, M. Kressel, H. E. Raybould, W. L. Neuhuber,
Vagal sensors in the rat duodenal mucosa: Distribution
and structure as revealed by in vivo DiI-tracing.
Anat. Embryol. (Berl.) 191, 203–212 (1995). doi: 10.1007/
BF00187819; pmid: 7771683
21. L. R. Beutler et al., Dynamics of gut-brain communication
underlying hunger. Neuron 96, 461–475.e5 (2017).
doi: 10.1016/j.neuron.2017.09.043; pmid: 29024666
22. Z. Su, A. L. Alhadeff, J. N. Betley, Nutritive, post-ingestive
signals are the primary regulators of AgRP neuron activity.
Cell Reports 21, 2724–2736 (2017). doi: 10.1016/
j.celrep.2017.11.036; pmid: 29212021
23. C. Brandon, D. M. Lam, L-glutamic acid: A neurotransmitter
candidate for cone photoreceptors in human and rat
retinas. Proc. Natl. Acad. Sci. U.S.A. 80, 5117–5121 (1983).
doi: 10.1073/pnas.80.16.5117; pmid: 6136039
24. O. P. Ottersen et al., Molecular organization of a type of
peripheral glutamate synapse: The afferent synapses
of hair cells in the inner ear. Prog. Neurobiol. 54,
127–148 (1998). doi: 10.1016/S0301-0082(97)00054-3;
pmid: 9481795
25. H. Haeberle et al., Molecular profiling reveals synaptic
release machinery in Merkel cells. Proc. Natl. Acad. Sci. U.S.A.
101, 14503–14508 (2004). doi: 10.1073/pnas.0406308101;
pmid: 15448211
26. D. A. Berkowicz, P. Q. Trombley, G. M. Shepherd, Evidence for
glutamate as the olfactory receptor cell neurotransmitter.
J. Neurophysiol. 71, 2557–2561 (1994). doi: 10.1152/
jn.1994.71.6.2557; pmid: 7931535
27. J. S. Marvin et al., An optimized fluorescent probe for
visualizing glutamate neurotransmission. Nat. Methods 10,
162–170 (2013). doi: 10.1038/nmeth.2333; pmid: 23314171
28. I. R. Wickersham et al., Monosynaptic restriction of transsynaptic
tracing from single, genetically targeted neurons. Neuron 53, 639–647
(2007). doi: 10.1016/j.neuron.2007.01.033; pmid: 17329205
29. T. Sato et al., Single Lgr5 stem cells build crypt-villus
structures in vitro without a mesenchymal niche. Nature 459,
262–265 (2009). doi: 10.1038/nature07935; pmid: 19329995
AC KNOWLED GME NTS
The authors wish to thank Y. H. Kim, A. Chamessian, M. Park,
Downloaded from http://science.sciencemag.org/ on September 20, 2018
S. Swain, M. Foster, C. Chen, W. Liu, and G. Wen for their
contributions. We also thank A. Pereda, R. Liddle, S. Lisberger,
E. Bohórquez, and the Grass Laboratory and Grass Fellows classes
of ’14, ’16, and ’17 for their constructive feedback. We thank
L. Looger for his gracious donation of the iGluSnfR plasmid. Our
sincere appreciation is expressed to the staff of the Duke Light
Microscopy and Flow Cytometry Core Facilities. The laboratory
mailing address is MSRB-I, room 221A, 203 Research Drive,
Durham, NC 27710, USA. Funding: NIH K01 DK-103832 (PI,
D.V.B.), NIH R03 DK114500-01 (PI, D.V.B.), AGA-Elsevier Pilot
Research Award (PI, D.V.B.), NIH P30 DK034987 UNC-CGIBD
Pilot Research Award (PI, D.V.B.), DARPA-ElectRx N2002850300
(PI, X.S.; Co-I, D.V.B.), NIH 1OT2OD023849-01 (PI, X.S.; Co-I,
D.V.B.), The Hartwell Foundation (PI, D.V.B.), The Dana Foundation
(PI, D.V.B.), and The Grass Foundation (PI, D.V.B.). K.L.B. is a
Howard Hughes Medical Institute Medical Research Fellow.
Author contributions: M.M.K. planned animal breedings and
qPCR experiments and performed monosynaptic rabies tracing,
electrophysiology experiments, calcium imaging recordings,
immunohistochemistry, and data analysis. M.M.K., K.L.B., and
B.B.B. optimized vagal cuff protocol and initial data analysis. K.L.B.
performed all vagal cuff recordings, immunohistochemistry, and
data analysis. M.E.K. manufactured monosynaptic rabies strains,
culture organoids, rabies infection of enteroendocrine cells in
organoids, organoid-nodose monosynaptic tracing, and data analysis.
M.M.M. performed all animal breeding, mouse colony management,
genotyping, and quality control. X.S. advised with biomedical
implants, including abdominal window, as well as data analysis. M.M.K.
and D.V.B. planned experiments and composed figures. D.V.B.
conceptualized, acquired funding for, and supervised the project,
as well as wrote the final manuscript. Competing interests:
Some of the findings in this manuscript have been used to file a
provisional patent application. No other competing interests are
declared. Data and materials availability: All data are available in
the manuscript or the supplementary materials.
SUPPLEMENTARY MATERIALS
www.sciencemag.org/content/361/6408/eaat5236/suppl/DC1
Materials and Methods
Figs. S1 to 20
Table S1
References (30–37)
Movies S1 and S2
Data S1
12 March 2018; accepted 2 August 2018
10.1126/science.aat5236
8 of 8
A gut-brain neural circuit for nutrient sensory transduction
Melanie Maya Kaelberer, Kelly L. Buchanan, Marguerita E. Klein, Bradley B. Barth, Marcia M. Montoya, Xiling Shen and Diego
V. Bohórquez

Science 361 (6408), eaat5236.

DOI: 10.1126/science.aat5236

Dissecting the gut-brain axis

It is generally believed that cells in the gut transduce sensory information through the paracrine action of
hormones. Kaelberer et al. found that, in addition to the well-described classical paracrine transduction, enteroendocrine

Downloaded from http://science.sciencemag.org/ on September 20, 2018

cells also form fast, excitatory synapses with vagal afferents (see the Perspective by Hoffman and Lumpkin). This more
direct circuit for gut-brain signaling uses glutamate as a neurotransmitter. Thus, sensory cues that stimulate the gut could
potentially be manipulated to influence specific brain functions and behavior, including those linked to food choices.
Science, this issue p. eaat5236; see also p. 1203

ARTICLE TOOLS http://science.sciencemag.org/content/361/6408/eaat5236

SUPPLEMENTARY http://science.sciencemag.org/content/suppl/2018/09/19/361.6408.eaat5236.DC1
MATERIALS

RELATED http://science.sciencemag.org/content/sci/361/6408/1203.full
CONTENT

REFERENCES This article cites 35 articles, 5 of which you can access for free
http://science.sciencemag.org/content/361/6408/eaat5236#BIBL

PERMISSIONS http://www.sciencemag.org/help/reprints-and-permissions

Use of this article is subject to the Terms of Service

Science (print ISSN 0036-8075; online ISSN 1095-9203) is published by the American Association for the Advancement of
Science, 1200 New York Avenue NW, Washington, DC 20005. 2017 © The Authors, some rights reserved; exclusive
licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. The title
Science is a registered trademark of AAAS.